Sanglard D, Ischer F, Monod M, Bille J
Institut de Microbiologie, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.
Antimicrob Agents Chemother. 1996 Oct;40(10):2300-5. doi: 10.1128/AAC.40.10.2300.
Some Candida albicans isolates from AIDS patients with oropharyngeal candidiasis are becoming resistant to the azole antifungal agent fluconazole after prolonged treatment with this compound. Most of the C. albicans isolates resistant to fluconazole fail to accumulate this antifungal agent, and this has been considered a cause of resistance. This phenomenon was shown to be linked to an increase in the amounts of mRNA of a C. albicans ABC (ATP-binding cassette) transporter gene called CDR1 and of a gene conferring benomyl resistance (BENr), the product of which belongs to the class of major facilitator multidrug efflux transporters (D. Sanglard, K. Kuchler, F. Ischer, J. L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother. 39:2378-2386, 1995). To analyze the roles of these multidrug transporters in the efflux of azole antifungal agents, we constructed C. albicans mutants with single and double deletion mutations of the corresponding genes. The mutants were tested for their susceptibilities to these antifungal agents. Our results indicated that the delta cdr1 C. albicans mutant was hypersusceptible to the azole derivatives fluconazole, itraconazole, and ketoconazole, thus showing that the ABC transporter Cdr1 can use these compounds as substrates. The delta cdr1 mutant was also hypersusceptible to other antifungal agents (terbinafine and amorolfine) and to different metabolic inhibitors (cycloheximide, brefeldin A, and fluphenazine). The same mutant was slightly more susceptible than the wild type to nocodazole, cerulenin, and crystal violet but not to amphotericin B, nikkomycin Z, flucytosine, or pradimicin. In contrast, the delta ben mutant was rendered more susceptible only to the mutagen 4-nitroquinoline-N-oxide. However, this mutation increased the susceptibilities of the cells to cycloheximide and cerulenin when the mutation was constructed in a delta cdr1 background. The assay used in the present study could be implemented with new antifungal agents and is a powerful tool for assigning these substances as putative substrates of multidrug transporters.
一些来自患有口腔念珠菌病的艾滋病患者的白色念珠菌分离株在长期使用唑类抗真菌药氟康唑治疗后，正变得对该化合物耐药。大多数对氟康唑耐药的白色念珠菌分离株无法积累这种抗真菌药，这被认为是耐药的一个原因。已表明这种现象与一种名为CDR1的白色念珠菌ABC（ATP结合盒）转运蛋白基因以及一种赋予苯菌灵耐药性（BENr）的基因的mRNA量增加有关，其产物属于主要易化子多药外排转运蛋白类别（D. Sanglard、K. Kuchler、F. Ischer、J. L. Pagani、M. Monod和J. Bille，《抗菌药物化疗》39:2378 - 2386，1995）。为了分析这些多药转运蛋白在唑类抗真菌药外排中的作用，我们构建了相应基因单缺失和双缺失突变的白色念珠菌突变体。测试了这些突变体对这些抗真菌药的敏感性。我们的结果表明，Δcdr1白色念珠菌突变体对唑类衍生物氟康唑、伊曲康唑和酮康唑高度敏感，因此表明ABC转运蛋白Cdr1可以将这些化合物用作底物。Δcdr1突变体对其他抗真菌药（特比萘芬和阿莫罗芬）以及不同的代谢抑制剂（放线菌酮、布雷菲德菌素A和氟奋乃静）也高度敏感。同一突变体对诺考达唑、浅蓝菌素和结晶紫的敏感性略高于野生型，但对两性霉素B、尼克霉素Z、氟胞嘧啶或普拉地米星不敏感。相比之下，Δben突变体仅对诱变剂4 - 硝基喹啉 - N - 氧化物更敏感。然而，当在Δcdr1背景下构建该突变时，此突变增加了细胞对放线菌酮和浅蓝菌素的敏感性。本研究中使用的检测方法可用于新的抗真菌药，并且是将这些物质指定为多药转运蛋白推定底物的有力工具。
